Engineering RNA editing tools for the generation of functional tRNA-derived small RNAs in the kidney

用于在肾脏中生成功能性 tRNA 衍生小 RNA 的工程 RNA 编辑工具

基本信息

批准号：
10751516
负责人：
JOSEPH VINCENT BONVENTRE
金额：
$ 33.18万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-20 至 2025-09-19
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10751516
关键词：
Acceleration Acute Acute Renal Failure with Renal Papillary Necrosis Alanine-Specific tRNA Animals Area Attenuated Autophagocytosis Binding Proteins Biogenesis Biological Assay Biological Process Biology Cell Death Cells Cellular Stress Chimeric Proteins Chronic Kidney Failure Clustered Regularly Interspaced Short Palindromic Repeats Communities Conserved Sequence Cysteine-Specific tRNA Data Disease Disease Progression Disease model Engineering Future Gene Silencing Generations Genetic Transcription Genome Goals Guanine Guide RNA Histology Human In Vitro Injections Injury to Kidney Kidney Kidney Diseases Messenger RNA Modeling Modification Mus Names Northern Blotting Oligonucleotides Organ Outcome Parents Pathology Phase Play Process RNA RNA Editing RNA-targeting therapy Regulation Reporting Resource Sharing Ribonucleases Ribosomes Role Serotyping Small RNA Stress Structure System Techniques Testing Therapeutic Transcript Transfer RNA Western Blotting adeno-associated viral vector angiogenin base cell type delivery vehicle design epigenetic regulation gain of function in vivo in vivo Model innovation interest kidney cell knock-down mRNA Stability mouse model preservation prevent promoter response stress granule stressor therapeutic RNA tool transcriptome transcriptome sequencing viral gene delivery

项目摘要

1 tRNAs have been recently demonstrated to be processed by ribonucleases into smaller regulatory fragments 2 named tRNA-derived small RNAs (tDRs) with their own distinct function including mRNA stability and silencing, 3 stress granule formation and epigenetic regulation. The function of these tDRs are dependent on their 4 sequence conservation and base modification that in turn determine their structure and protein-binding ability. 5 We have previously detailed the dynamic regulation of tDRs in response to cellular stress and have in the 6 process identified Asp-GTC-3’tDR that plays a critical role in stress-dependent induction of autophagy, is highly 7 expressed in the kidney and appears to be reno-protective in vitro. In several murine in vivo models, cellular 8 levels of Asp-GTC-3’tDR initially increases in the acute phase of renal injury, and subsequently decreases in 9 chronic kidney disease models. Notably in vivo silencing of Asp-GTC-3’tDR in the acute phase of renal injury 10 accelerates cell death and disease progression suggesting a compensatory response. However, tools to 11 manipulate the expression of this and other tDRs in vivo that preserve their cellular modifications presents a 12 significant hurdle in the field. Our preliminary data suggest that the RNA-targeting CRISPR/Cas13, guided by 13 gRNAs, can induce programmable cleavage(s) on tRNAs and generate functional tDRs without disturbing the 14 parent tRNA pool. Notably, we built a functional and smaller Cas13/ANG chimeric protein by replacing two 15 HEPN domains of pspCas13b with Angiogenin (ANG), a small RNase responsible for tDR biogenesis that 16 permits packaging into viral gene delivery vectors. The long-term objective of this proposal is to is to develop a 17 programmable Cas13/ANG platform packaged in AAVs for delivery to kidneys for the biogenesis of 18 endogenous functional tDRs. To achieve our objectives, we plan to: 1) Test the hypothesis that the engineered 19 CAS13/ANG platform, customized with suitable gRNAs, generates endogenous functional tDRs. As a proof-of- 20 concept, we focus on three tDRs with clear readouts: stress granule-inducing Ala-AGC-5’tDRs and Cys-GCA- 21 5’tDRs, and autophagy-inducing Asp-GTC-3’tDRs. These will be tested in HEK cells for their ability to induce 22 robust tDR generation without disturbing the parent tRNA pools; assays checking for stress granule formation 23 and autophagy flux will be used to evaluate the functionality of Cas13/ANG-generated tDRs; and 2) Use AAV- 24 delivered Cas13/ANG machinery to generate functional Asp-GTC-3’tDR in kidneys and determine if this 25 attenuates CKD progression in our mouse CKD model. The functional effects of induction of Asp-GTC-3’tDR 26 will be assessed by northern blotting, histology, immunostaining, western blotting, and RNA-seq to determine 27 effects on autophagy and the progression of CKD. We expect this platform which we aim to make widely 28 available to the scientific community could be used in many different systems to both study the function of 29 tDRs in vivo, and also provide a possible therapeutic avenue for tDR-based therapeutics.

1 最近已证明 tRNA 可被核糖核酸酶加工成更小的调节片段 2 个名为 tRNA 衍生的小 RNA (tDR) 具有自己独特的功能，包括 mRNA 稳定性和沉默， 3 应激颗粒的形成和表观遗传调控这些 tDR 的功能取决于它们的功能。 4 序列保守和碱基修饰反过来决定了它们的结构和蛋白质结合能力。 5 我们之前详细介绍了 tDR 响应细胞应激的动态调节，并在 6个过程确定了Asp-GTC-3’tDR在应激依赖性自噬诱导中发挥着关键作用，高度 7 在肾脏中表达，在体外细胞模型中似乎具有肾保护作用。 8 Asp-GTC-3’tDR 水平最初在肾损伤急性期升高，随后降低 9 慢性肾病模型，尤其是肾损伤急性期的 Asp-GTC-3’tDR 体内沉默。 10 加速细胞死亡和疾病进展，表明存在补偿反应。 11 在体内操纵该 tDR 和其他 tDR 的表达，以保留其细胞修饰，呈现出 12 该领域的重大障碍我们的初步数据表明，RNA 靶向 CRISPR/Cas13 在指导下。 13 gRNA，可以诱导 tRNA 上的可编程切割并生成功能性 tDR，而不会干扰值得注意的是，我们通过替换两个亲本 tRNA 库构建了一个功能性且较小的 Cas13/ANG 嵌合蛋白。 pspCas13b 的 15 个 HEPN 结构域与血管生成素 (ANG)，这是一种负责 tDR 生物合成的小型 RNase， 16 包装许可进入病毒基因传递载体该提案的长期目标是开发一种 17 个可编程 Cas13/ANG 平台封装在 AAV 中，用于递送至肾脏以进行生物合成 18 种内源性功能 tDR 为了实现我们的目标，我们计划： 1) 检验工程化的假设。 19 使用合适的 gRNA 定制的 CAS13/ANG 平台可生成内源性功能 tDR 作为证明。 20 概念中，我们重点关注具有清晰读数的三种 tDR：应激颗粒诱导的 Ala-AGC-5’tDR 和 Cys-GCA- 21 5’tDR 和自噬诱导 Asp-GTC-3’tDR 将在 HEK 细胞中测试其诱导能力。 22 种稳健的 tDR 生成，无需干扰亲代 tRNA 池即可检查应激颗粒的形成； 23 和自噬通量将用于评估 Cas13/ANG 生成的 tDR 的功能；以及 2) 使用 AAV- 24 交付 Cas13/ANG 机器以在肾脏中生成功能性 Asp-GTC-3’tDR 并确定这是否 25 在我们的小鼠 CKD 模型中减弱 CKD 进展 Asp-GTC-3’tDR 诱导的功能作用。 26 将通过 Northern blotting、组织学、免疫染色、western blotting 和 RNA-seq 进行评估，以确定 27 对自噬和 CKD 进展的影响我们期望我们的目标是广泛推广这个平台。 28 科学界可用的可用于许多不同的系统，以研究 29 体内 tDR，也为基于 tDR 的疗法提供了可能的治疗途径。